Construction and Characterization of a Functional GLIC-GABAρ Ligand-Gated Ion Channel Chimera

Abstract

Pentameric ligand gated ion channels (pLGICs), especially anion-selective GABAARs, are major targets of general anesthetic (GA) drugs. GAs mediate their actions by binding in the transmembrane domain (TMD) of pLGICs, which potentiates anion-selective pLGIC currents and inhibits cation-selective pLGICs. Recently, crystal structures of prokaryotic cation-selective pLGIC homologs, GLIC and ELIC, have been solved with several GAs bound. High-resolution structures that reveal how GA's interact with anion-selective channels such as the GABAAR are not available, which justifies a quest for pLGIC constructs that preserve GA positive modulation and are amenable for structural studies.Here, we report the construction and characterization of a GLIC-GABAρ chimeric pLGIC that is positively modulated by the GA pentobarbital. We fused the extracellular domain (ECD) of the prokaryotic proton-gated homopentameric channel GLIC with the eukaryotic GABAρ subunit TMD. GABAAR ρ1 subunits form homopentameric channels making them ideal for constructing chimeras with GLIC. The GLIC-ρ chimeric construct formed functional proton-gated channels when expressed in Xenopus oocytes. The reversal potential of the chimeric channels was −31mV, which is similar to the reversal potential of GABAρ receptors suggesting that the channel is chloride-selective. GABAρ receptors are normally insensitive or inhibited by GAs but a point mutation in GABAρ TMD W328M converts it to a channel that is positively modulated by pentobarbital. When this mutation was made in the GLIC-GABAρ chimeric construct, the mutant chimeric channel was directly gated by pentobarbital and proton-mediated currents were potentiated by pentobarbital. Protocols for expressing and purifying high levels of the GLIC-GABAρ chimeric channel protein are currently being developed.